Process for preparing l-glutamic acid



United States 1 Patent Ofitice PROCESS FOR PREPARING L-GLUTAMIC ACID This invention relates to the manufacture of L-glutamic acid by fermentation, and more particularly to a carbon source for the fermentative preparation of glutamic acid.

Many microorganisms are knownto be capable of producing glutamic acid from suitable carbon and nitrogen 1 column lists the glutannc acid concentration produced in sources by fermentation. In typical industrially employed fermentation processes, glucoseis the carbon source. The nitrogen source may be any one or a mixture of ammonia, ammonium sulfate, ammonium nitrate, or urea. The

glutamic acid is produced when a wide variety of microorganisms is cultured aerobically at a temperature between about 27 C. and 33 C. -on a nutritive medium containing such carbon and nitrogensources togetherwith molasses contain about 50% sucrose and the several juices also are rich in sucrose. Attempts have previously been madeto employ the sucrose of molasses and of other raw and intermediate sugar products as a carbon source for the fermentative production of glutamic acid. These at- 1 tempts have failed to result in practical industrial processes for two primary reasons. Molasses and other sugars contain colored substances which'inhibit the fermentative production of glutamic acid by microorganisms. They can be removed by adsorption on diatomaceous earth and the like. Molasses and other raw or intermediate sugar products also contain substantial amounts of biotin and of other substances which promote the growth of the microorganisms at the expense of their glutamic acid production. Therefore, in spite of low cost, molasses and other inexpensive sucrose containing materials have not heretofore been an economical carbon source for glutamic acid fermentation. 1

The amounts of growth promoting constituents in the several raw and intermediate products mentioned above vary but they are approximately equally effective with We have found that the metabolism of all microorganisms normally" employed for producing glutamic acid can be influenced by adding non-ionic surface active agents to culture media Whose carbon source is molasses or sucrose in other forms. Glutarnic acid can be produced inhigh yields and high concentration in culture m edia which containmolasses, or other sources of sucrose and surface active agents. I

,The following table lists the results of experiments in which several surface activeagents were addedto culture media containing10 g./dl. of sucrose in the form of beet molasses, 0.1 g./dl. of KH PO 0.04 g./dl. of

V MgSO4.7I -I 'O and 0:6 g./dl. of urea. The media were inoculated with Brevibacherium lact ofermentum, strain No. 2256 (ATCC No. 13869), and cultured at 30 C. for 48 hours, with shaking. The pH of the media was maintained in the alkaline range near pH 7.5 by adding ureasolution. The first column of the table lists trademarks of surface active agents used. The; seco nd column lists the initial surface active agent concentration in per cent, The third the media.

TABLE. 1

Surface-Active Glutamic Acid Surface Active Agent "Agent Concer- Concentration, r tration 8./ dl. 7 (Percent) Tween 20 is a polyoxyalkylene derivative of sorbitan monolaurate.

. Tween 40 is the corresponding derivative of sorbitan monopaimitate,

1 Tween 80 is the corresponding derivative of sorbitan monooleate.

Span 20 is sorbitan monolaurate.

Aldo 28 is giyceryl monostearate. i The eifect of the'surface activeagents'on the growth of glutamic acid producing microorganisms 'is illustratedin Table 2 which lists results of'tests performed on micro? organisms grown on media containing varying amounts of surface active agent. In. all tests, the culture medium contained 10 g./dl. molasses, 0.1 g./dl. KH PO 0.04 g./dl. MgSO ;7H O, 2 p.p.m',: Fe ions. 2 ppm. Mn ions, 0.8. g'.'/dl. urea,.5'y/1. biotin, and 0.5 ml./dl. soybean protein hydrolyzate. Themicroorganism employed was Brevibact erium lactofermentum,*strain No. 2256 (ATC C No. 13869). After. 42 hours incubation, the bacterial growth was determined by measuring theabsorbency of the culture'medium ata wavelength of 562 millimicrons and the residual sucrose was measured. i

TABLE 2 V Residual I Turbidity sucrose Surface Active Agent Concentration (Percent) oteulture Concentration,

g./dl

. NOTE.Sp8.n 60 is sorbitanmonostearate. Tweeri'fioris the po1yoxyalkylene derivative of sorbitan monostearate.

It is evident from Table 2 that the surface active agents Patented Jan. 5, 1965 donot inhibit the normal 7 V glutamie acid. When a large excess of surface act ve' if Surface Active ice box, 335 g. of crystallized crude L-glut a I growth of bacterial producing agent'is added to thejmedium, the microorganisms cannot metabolize sucrose to produce glutamic acid, as' will become'further evident from Table 3.- 1 Addition of surface active agents in moderate amounts; unfavorably affects the proliferation of microorganisms -relative to the glutamic acid produced. 1

Therefore, the metabolism of microorganisms producing glutamic acid can'b'e. controlled to produce glutarnic acid in high yield by addition of surface active agents in amounts smaller than those which inactivate the glutamic acid. producing enzyme in the microbialcells;

7 Table 3 shows that an excessiveor an insufiicient initial concentration of surface active agent reduces the yield.

The cultureme'clium in each test contained g./dl. beet -m'olasses, 0.1 g./dl. KH PO' 0.04 g./dl.'-MgSO .'IH O, and 0.6 g./d1. urea. The surface active agent used was proceeded for 48 hours at Tween 60. The incubation 30 C. with shaking. TABLE 3 Agent Concentration Residual Percent) v I Sugar (g./dl.) Produced "(s-l l 5 9 9 5 999 61- commune orw oamsomm i The following detailed examples further illustrate the fermentative manufacture of L-glutamic' acid in the presence of surface active agents from culture media con;

Glutamic Acid p of 'Brevzbacterzum, lactofermentus,

- Example 1. g

-Brevibacterium flavum, strain INo. 2247 (ATCC No.

14069) The fermentation was permitted'to proceed at 31 C. for 42 hours undershaking. A 45% aqueous solution of urea was added to each flask during fermentation to keep the culture mediumslighlty alkaline. From each liter of saidbroth 28 grams of crystallized crude L glutamic acid were obtained in the manner described in Ten litersof an aqueous solution of beet molasses containing 47 percent' sucrose were diluted with an equal volume of'wa-tcrwiththorough stirring. 500 grams of diatom aceous earth were added tothe diluted solution, which was thereafter filtered with suction. A culture medium was prepared from 6.6 litersof the filtrate, vl4

grams of potassium dihydrogen phosphate, 5.6 grams .of

magnesium sulfate heptahydrate and 14 grams of Tween 60. The pHbf the medium was adjusted to 7.0 with ammonium hydroxide. It was diluted to 13.3liters and placed in a liter stainless steel fermentation jar. The medium in the jar was sterilized at 115 C. for 20 minutes, cooled, and inoculated with 700 m:l.of a seed liquor 7 strain No. 2256 (ATCC No... 13869),which had been cultured 15 hours. Fermentation was carried out in the jar of 31 C. with aeration and agitation. Duning ferinentation, gaseous ammonia was introduced into the jar together with the air to keep the pH value'of theculture medium at approxi- .30

mately 7.8, 48 hours after inoculation, fermentation was completed, and the concentration of L-glutamic acid in the fermentation broth was 4.38 grams .per deciliter. The broth was filteredafter addition of 1.5 percent taining molasses or other forms'of sucrose as a carbon 7 source. 9

Example 1 Beet molasses containing 4.7 g.:/ dl. sucrose was diluted 7 with water to approximately'two times its initial volume. Diatomaceous earth was added to the diluted molasses which wasthen filtered. The filtrate was further diluted with water to a sucrose concentration-of 1O g./cll. A culture medium was prepared from this sucrose solution by additionof 0.1 ,.g./dl." potassium dihydro'gen phosphate, 0.04 g. '/dl. magnesium-sulfate heptahydrate, 0.6

of diatomaceous earth to remove the bacterial cells. The filtrate was decolorized by addition of activated carbon and filtration, and finally concentrated under reduced pressure. When the residue-lwas acidified to'pH 3.2 with hydrochloric acid and cooled'overnightin an ice box, 510 grams of crude L-glutaniieacid were precipitated and recovered by filtration.

A culture mediumcontaining l2 g./dl. sucrose in the form of sugar-beet molasses, 0.1 g./dl. diammonium hydrogen phosphate, 0.04 'g ./dl. magnesium sulfate heptahydrate and- 0.6 g./dl'.'urea, was prepared in the manner'described in Example 1. 20 ml. batches of the g./dl. urea'and 0.1 gL/dlzTWeen 60. The pH of the medium was adjusted to 7 .0 with aqueousammonia solu'-.

tion. 20 ml. batches of'the culture medium were placed in 500 ml. .flasks; sterilized at 120 C.-for 15 minutes, and inoculated ,with- Brevibacterium lactofermentus,

strain No, 225 6 (ATCC No..13'869), which had pre- The fermentation viously' been cultured. for 24. hours. was permitted to proceed at 31 Cpfor 48 hours under shaking. Anaqueous solution of urea was added to each flask during fermentation to keep the culturemedium slightly alkaline The: medium after fermentation contained 4.00 g./dl.' L-glutamic acid. The culture culture medium were placed in 500 ml. flasks, sterilized, and then inoculated with Brev'ilmcterium lactofermentus,

7 strain No. 2256 (ATCC No. 13 869) previously prepared by culturing for 24' hours.v The'fer mcntation was permitted to proceed at 31 C. under shaking. An aqueous solution of urea was added to each flask during fermentation to keep thecultureniedium' slightly alkaline. Six

hours after inoculation, the flasks were divided into two broth obtained after fermentation was filtered in order to remove the cells therefrom; concentrated under reduced pressure, and its pH was adjusted to the isoelectric point of glutamic acid.(3.2) by means of hydrochloric acid. When the adjusted broth was kept overnight in an amic acid were obtained from each. liter of the original broth.

' Example" V I 'fAn aqueous solution-of 10 g'./dl; sucrose prepared'in the'manner described in Example 1, was mixed'with 0.1 g./ d1. potassium dihydrogenph'osphate, 0,0 4 g./dl. mags nesium sulfate heptahydrate, 0.6 gQ/dl. urea,,a.nd 0.1 g./dl. Aldo 28. The pH of the culture medium so'obtained was adjusted to 7.0 with aqueous ammonia solution. 20 ml. batches of the culture medium were placed in 500 flasks, sterilized, and then inoculated with was 5.56. g./dl. in the groups. The contents of each flask in the first group Crystals of. raw beet sugar were dissolved in water to form 21209;, solution. Diatomaceous earth was added to the solution which was then filtered. The filtrate was further diluted with water to a sucrose concentration of 10 g./dl. A" culture medium was prepared from this sucrose solution by addition of 0.3'g.-/ dl. potassium dihydrogen phosphate,f0.04 g./ d1. magnesium sulfate hydrate, 0.6

g./dl. ureaarid 0.04 g./dl. Tween 60. The pH of the medium was adjusted .to'7 .0 with aqueous ammonia solu tion. 20 m1. batches of the culturemedium were placed.

in 500 flasks, sterilized at 120 C. for 15 minutes, and

during fermentation to keep the culture medium slightly alkaline. The medium after fermentationcontained 3.87 g./ d1. L-glutamic acid. ,The culture broth'obtained after fermentation was filtered in order to remove 'the cells, .con-

centrated under reduced pressurqand its pH was adjusted to the isoelectric point of glutamic acid,(3.2) by 'means of hydrochloric acid. When the adjusted broth was kept overnight in an ice box, 34.1 g.,of crystallized crude L- glutamic acid were obtained fromeach liter of the original broth. i V ,l,

. Example6 j Crystals of, raw cane were dissolved in water, and diatomaceous earthwas added to the solution which was then filtered. The filtrate was further diluted with water to a sucrose concentration of g./dl., anda culture medium was prepared from this solution by the addition of 0.2 g./dl. diammonium hydrogen phosphate, 0.04 g./dl. magnesium sulfate heptahydrate, 0.6 g./dl. urea and 0.03 g./ dl. Aldo 28. I The pHof the medium was adjusted to 7.0 with aqueous ammonia solution. theculture medium were placed in 500 ml. flasks, sterilized, and inoculated with Brevibacterium flavum, strain No. 2247 (ATCC No. 14069). The fermentation'was permitted to proceed at 31 C. for 42 hours under shaking. An aqueous solution of urea was added to each flask during'fermentation to keep the culture medium slightly alkaline. ,27' grams of, crystallized crude L-glutamic acid were obtained from each liter of the original broth in the manner describedin Example 1. 1

Example 7 L-glutamic acid fermentation was. carried out in the The fermentation was permitted to 1 medium w... prepared from the diluted solution by addition of 0.2% potassiumdihydrog'en phosphate, 0.04%

20 ml. batches of to the solution, which ,was then filtered with suction. A

culture medium was prepared from 6.6 liters of the filtrate, 28 grams potassium dihydrogen phosphate, 5.6 grams magnesium. sulfate heptahydrate and 4.2 grams Tween 60. The pH of the medium was adjusted to 7.0 .with aqueous ammonia. It was diluted to 13.3 liters and placed in a 20 liters stainless steel fermentation jar. The medium in the jar was sterilized at 115 C. for-20 minutes, cooled, and inoculated with 700 ml. of a seed liquor of Brevibacterium lactoferm enlus, strain No. 2256 (ATCC No. 13869), which had been cultured 15 hours. Fermentation was carried outin thejar at 31 C. with aeration and agitation. During fermentation, gaseous ammonia was introduced into the jar' together with air at such a rate that the pH value of the culture medium was kept at approximately 7.8. i 48" hours after inoculation, fermentation was terminated. The concentration of L-glutamic acid in the fermentation broth was 4.52 g./ d1. The broth was filtered after addition of 1.5 percent diatomaceous earth to remove the bacterial cells. The filtrate l 100 of raw sugar beet juice were diluted with water to adjust the sucrose concentration to 10%. A culture -was decolorized by the addition of aetivated carbon, and

magnesium sulfate heptahydrate and 0.05% Tween 40.

20 ml. batches of the culture medium were placed in 500 ml. flasks, sterilized at 120 C. for 20 minutes, and

the culture medium slightly alkaline. The mediumafter (fermentation contained 4.15 g./dl L-glutamic acid.

When the fermentation was carried out in the same manner but without Tween 40, the concentration of L-glutamic acid in the culture medium was 1.06 g./ d1.

7 Example 10 Blackstrap molasses was dissolved in water. Diatomaceous earth was added and the solution was filtered with suction. Aculture medium was prepared from 6.6 liters of the clarified solution, 14 grams potassium dihydrogen phosphate, 5.6 grams magnesium sulfate heptahydrate, and 56 grams Tween 40. The pH of the medium was adjusted to 7.0 with ammonia. It was diluted to 13.3 liters and placed in a'20 literstainless steel fermentation jar. 'The' medium inthe jar was sterilized at 115 C. for 20 minutes, cooled, and inoculated with 700 ml. of a seed liquor of Brevibacterium sacchgrolylicum (ATCC No. 14066), which had been cultured 15 hours. Fermentation was carried out in the jar at 31 C. with aeration and agitation. During fermentation gaseous ammonia was introduced into the jar togetherwith the air whereby the pH value, of the culture medium was kept at approximately 7.8. ,48 hours after inoculation, the fermentation was terminated. The concentration of L-glutamic acid in the fermentation broth'was 4.13 grams per deciliter.

. Example 11 Blackstra-p molasses was dissolved in water and diatomaceous earth was added to the solution which was then filtered with suction. A culture medium was prepared from 6.6 liters of the clarified solution, 14 grams potassium dihydrogen phosphate, 5.6 grams magnesium sulfate heptahydrate. The pH of medium was adjusted to 7.0 with ammonium hydroxide. It was diluted to 13.3 liters and placed in a 20 liters stainless steel fermentation jar. The medium in the jar was sterilized at 115 C. for 20 minutes,.-cooled and inoculated with 700 ml. of a seed liquor of Brevibacterium divaricatum (NRRL B-23 12.), which hadbeen cultured 15 hours. Fermentation was carried out in the jar at 31 C. with aeration and agitation. During fermentation, gaseous ammonia was introduced into the jar together with the air so that the pH value of the culture medium was kept at approximately 7.8. When the growth of microorganism had raised the light absorbency (turbidity) to 0.32 five hours after inoculation, 84- grarns glycerylmonostearate were added to the medium and fermentation was continued for 54 hours. The concentration of L-glutamic acid in fermentation liquid was 3.97 grams per deciliter.

Example 12 sterilized at l 15 C. for 20 minutes, cooled and inoculated continued for 42 hours. 645

monia was introduced into the jar together 60 grams Tween60 were added and fermentation was were collected from the broth. v a 1 Example '13 j "i Crystals of 'raw cane sugar were dissolved in water- Diatomaceous earth was addedto the solution which was then filtered, The filtrate was further diluted with water to a sucrose concentration of 10. g./dl. A culture medium was prepared from this solution by the addition of O.2 g./dl. diammonium hydrogen phosphate, 0.04 g./dl. magwith the air grams of L-glutamic acid ncsium sulfate heptahydrate, 0.6 g./dl. urea and 0.3 g./ dl. j

mixture of sucrose stearates (Nitto Ester-1009; brand n ame)-,a surface active agent. The pH of the medium I was adjusted to 7.0 with aqueous ammonia solution.

ml. batches of the culture medium were placed in- 500 ml. flasks, sterilized and inoculated with Brevibacterz'um,

immariophillium (ATCC No.140 68). The fermentation was permitted to proceed at 2830 C. for.42 hours under shaking. 'An aqueous solution of urea was added to each flask during fermentation to keep the culture medium at pH 7.58.2.-' The concentration of L-glutarnic acid in the" fermentation broth'was 3.24 g./dl. I

I E'lrample 14 Beet molasses containing 47 g./dl. sucrose was diluted with water to approximately two times its initial volume.

Diatomaceous earth was added tothe diluted molasses which was then filtered. The filtrate was further diluted were addedtofermentation medium. Fermentation was whereby the pI-I valueof vtheculture medium wasv kept I at'approxirnately 7.8. When the growth of microorganism I caused the absorbency of the medium to rise to 0.27,

continued for 52 hours. The ultimate concentration of L- gl-utamic acid in the fermentation liquidwas 4.31 g./dl.

I Example 16 I V 120 C. for 15 minutes, and innoculated with Brevl'ba'm tel-tum IactOfermentuS, j strain No. 2256 (A'ITC No. 13869), which had previously been cultured for 24 hours. The fermentation was permitted to proceed at-33 C. under shakin'g. An .aqeous 45% solution of .urea was addedto each flask during fermentation at arate sufiicietn' to keep the culture medium at pH 7.09,; -5.5 hours after inoculation, the grow-tlifof microorganism increased the'light absorbency of the medium to 0.28, and 0.44

- g./dl. polyo ryethylene oetadecyl ether, a surface active agent, were addedto the fermentation medium. Fermentation was continued for 44'hours.

was terminated,- the concentration of L-glutamic acid in lactofermentus, strain No. 2256 (ATCC No. 13869),

which had previously been cultured for 24 hours. The fermentation was permitted to proceed at 37 C. under shaking. An aqueous 45% solution of urea was added to eachfiask during fermentation to keep the culture medium slightly alkaline. 6.5- hours after inoculation the growth: of the microorganism had raised the. light absorbence to 0.40. At that time, 0.13 g./dl. Tween 60 were added to the fermentation medium, and fermentation was continued for 40 hours. The final concentration fermentation liquid was v of the L-glutamic acid in the 4.89 g./dl.

Example 15 Beet molasses containing 47 g./dl. sucrose was diluted with water to approximately two times-its initial volume.

13869), which had previously been cultured for 24 hours.

The fermentation was permitted to proceed at 30 C.

When fermentation the fermentation liquid was 3.98 g./dl.

' Example 17 Beet molasses containing 47 g./dl. sucrose was diluted with water to approximately two timesits initial volume. Diatomaceous earth was addedtothe diluted molasses which was then filtered. The filtrate was further diluted with water to a sucrose concentration of l0 g./d1. A

. culture medium was prepared from this solution by the addition of 0.1 g./ dl. potassium dihydrogen phosphate, 0.04 g./dl. magnesium sulfate'heptahydrate, and 0.6 g. d1. urea. The pH of the medium was adjusted to 7.0 with aqueous ammonia solution. 20 ml. batches of the culture medium were placed in 500 m1. flasks, sterilized at 120 C. for 15 minutes, and inoculated with Brevibacterium lactofermentus, strain No. 2256 (ATCC No. 13869), which had previously been cultured for 24 hours. The fermentation was permitted to proceed at 30 C. under shaking. An aqueous 45 solution of urea was added to each flask during fermentation to keep the culture medium at. pH 7.7. hours after inoculation the growth of the microorganisms had raisedfthe light absorbency to 0.30

' and 0.10 g./dl. poly'okyethylene' stearate (Nonion S4;

liquid was '4.77"g./dl. I

trademark) were added to fermentation medium. Fermentation was continued for 48 hours. The ultimate concentration of L-glutamic acid in the fermentation While the invention has been described with particular reference to specific embodiments, it is to be understood that it is not'lim'ited thereto but 'is' to be construed broadly and restricted solely by the scope of the appendedclaims.

What we claim is: l. A process for preparing L -glutamic acid which comprises culturing a microorganism capable of producing 'L-glutamic acid from a nitrogen source and from sucrose as .a carbon source on a medium containing ass carbon source a material selected from the group consisting of 7 molasses, raw juice," and raw sugar derived from sugar under-shaking. An aqueous 45 %-.solution of urea was;

added to'e'ach flask during fermentation to keep the cul: ture medium at'pH 7.7. 7 hours after inoculation the growth of the microorganisms had raised the light absorbancy to 0.41 and 0.12 g./dl. polyoxyethylene :palmitate beetsandsugar c'ane, said medium further containing an effective amount of a non-ionic surface active agent smaller than the amo'unt'of said surface active agent which face active agent -is a polyoxyethylene derivative of a inactivates the glutamicacid-producing enzymein said monoester of sorbitan with an acid selected from the group consisting of lauric, palmitic, oleic, and stearic acid.

3. A process as set forth in claim 1, wherein said surface active agent is a monoester of sorbitan with a monocarboxylic acid having a straight carbon chain of 12 to 18 carbon atoms.

4. A process as set forth in claim 1, wherein said surface active agent is glyceryl monostearate.

5. A process as set forth in claim 1, wherein said surface active agent is an ester of a polyhydric aliphatic alcohol with a monocarboxylic acid having a straight carbon chain of 12 to 18 carbon atoms, said alcohol in said ester having free hydroxyl groups.

6. A process as set forth in claim 1, wherein said surface active agent is sucrose stearate.

7. A process as set forth in claim 1, wherein said surface active agent is polyoxyethylene palmitate. I

8. A process as set forth in claim 1, wherein said surface active agent is polyoxyethylene octadecyl ether.

9. A process as set forth in claim 1, wherein said surface active agent is polyoxyethylene stearate.,

10. A process asset forth in claim 1, wherein said microorganism is a Brevibacterium capable of producing L-glutamic acid.

References Cited in the file of this patent UNITED STATES PATENTS 3,003,925 Kinoshita et a1 Oct. 10, 1961 3,036,958 Asai et al May 29, 1962 3,063,914 Polnitz et a1. Nov. 13, 1962 

1. A PROCESS FOR PREPARING L-GLUTAMIC ACID WHICH COMPRISES CULTURING A MICROORGANISM CAPABLE OF PRODUCING L-GLUTAMIC ACID FROM A NITROGEN SOURCE AND FROM SUCROSE AS A CARBON SOURCE ON A MEDIUM CONTAINING AS A CARBON SOURCE A MATERIAL SELECTED FROM THE GROUP CONSISTING OF MOLASSES, RAW JUICE, AND RAW SUGAR DERIVED FROM SUGAR BEETS AND SUGAR CANE, SAID MEDIUM FURTHER CONTAINING AN EFFECTIVE AMOUNT OF A NON-IONIC SURFACE ACTIVE AGENT SMALLER THAN THE AMOUNT OF SAID SURFACE ACTUVE AGENT WHICH INACTIVATES THE GLUTAMIC ACID PRODUCING ENZYME IN SAID MICROORGANISM. 